Abstract
Cancerous tissues undergo extensive changes to their cellular environments that differentiate them from healthy tissues. These changes include changes in extracellular pH and Ca(2+) concentrations, and the exposure of phosphatidylserine (PS) to the extracellular environment, which can modulate the interaction of peptides and proteins with the plasma membrane. Deciphering the molecular mechanisms of such interactions is critical for advancing the knowledge-based design of cancer-targeting molecular tools, such as pH-low insertion peptide (pHLIP). Here, we explore the effects of PS, Ca(2+) , and peptide protonation state on the interactions of pHLIP with lipid membranes. Cellular studies demonstrate that exposed PS on the plasma membrane promotes pHLIP targeting. The magnitude of this effect is dependent on extracellular Ca(2+) concentration, indicating that divalent cations play an important role in pHLIP targeting in vivo. The targeting mechanism is further explored with a combination of fluorescence and circular dichroism experiments in model membranes and microsecond-timescale all-atom molecular dynamics simulations. Our results demonstrate that Ca(2+) is engaged in coupling peptide-lipid interactions in the unprotonated transmembrane conformation of pHLIP. The simulations reveal that while the pH-induced insertion leads to a strong depletion of PS around pHLIP, the Ca(2+) -induced insertion has the opposite effect. Thus, extracellular levels of Ca(2+) are crucial to linking cellular changes in membrane lipid composition with the selective targeting and insertion of pHLIP. The characterized Ca(2+) -dependent coupling between pHLIP sidechains and PS provides atomistic insights into the general mechanism for lipid-coupled regulation of protein-membrane insertion by divalent cations.